Sequencing of the human and other genomes has radically changed the ways in which we identify and characterize genes. Typically, database searches that are based on structural similarities can assign a gene product to an established protein family. Although members of protein families often share a common mechanism of action, the cellular processes in which they are involved can be both highly specialized and fundamentally important.
Protein tyrosine phosphatases (PTPs), enzymes which have conserved catalytic domains but are involved in controlling a broad constellation of cellular processes (1-3), provide a striking example of conserved structures associated with functional diversity. The hallmark that defines the PTP superfamily is the active site amino acid sequence (H/V)C(X)5R(S/T) (SEQ ID NO:1), also referred to as the PTP signature motif, within the catalytic domain. To date, analysis of the nearly completed human genome has revealed 112 predicted human PTPs (4). Therefore, it is relatively easy to attribute a general role to a PTP gene product based upon structural homologies. However, determination of the exact physiological function of a PTP requires a tedious and protracted effort. Identification of the cellular substrates of individual PTPs will help to elucidate the biological functions of individual PTPs. A major challenge, though, is the development of technologies for rapid substrate identification that can be applied to the entire PTP family.
Detailed mechanistic studies have shown that PTPs utilize a common mechanism for phosphomonoester hydrolysis (5). See FIG. 1. The PTPs employ the active site cysteine (e.g., Cys215 in PTP1B) as the attacking nucleophile, thereby forming a thiophosphoryl enzyme intermediate (“E-P”) (6, 7). The E-P formation is assisted by a conserved aspartic acid (e.g., Asp181 in PTP1B), functioning as a general acid, to neutralize the build-up of a negative charge on the leaving group (8, 9). For the hydrolysis of E-P, Asp181, previously functioning as a general acid in E-P formation, acts as a general base, abstracting a proton from the attacking water (10, 11). This enhances the rate of E-P hydrolysis, thereby regenerating the active enzyme. The PTPs further accelerate the formation and hydrolysis of E-P by preferentially binding the pentacoordinated transition states with the guanidinium side chain of the active site arginine residue (e.g., Arg221 in PTP1B) (12, 13).
Because of the transient nature of the enzyme•substrate complex, it has been difficult to isolate substrates with wild-type PTPs. Based upon insights from mechanistic studies, two types of “substrate-trapping” mutant PTPs have been developed. In the first, the active site Cys residue is replaced by a Ser (14-16); in the second, the general acid Asp residue is substituted by an Ala (17, 18; see U.S. Pat. Nos. 5,912,138 and 5,951,979). These mutants retain the ability to bind substrates; however, because they are either unable to carry out substrate dephosphorylation (the Cys-to-Ser mutant) or severely impaired in carrying out substrate dephosphorylation (the Asp-to-Ala mutant), capture of the PTP enzyme•substrate complex becomes possible.
The substrate-trapping mutant PTPs have been used as affinity reagents to isolate and identify physiological substrates for various PTPs. Nevertheless, to date, only a limited number of PTP substrates have been identified by the substrate-trapping approach, and these have been mostly abundant proteins. For example, the adapter protein p130 has been found to be the target of several PTPs, including PTP1B (19), PTP-PEST (17), the Yersinia PTP (20), PTPα (21), LAR (22), and SAP (23). The fact that only a few proteins have been identified as PTP substrates is surprising, given the large number of protein tyrosine kinases and phosphotyrosine-(pTyr-) containing proteins in the cell. One possible explanation may be that the affinity of the available trapping mutants is not sufficiently high, such that only heavily populated phosphoproteins can be isolated. Accordingly, in view of the foregoing, there exists a need to create an improved PTP substrate-trapping mutant, with a higher affinity, that will enable the identification of less abundant substrates.